The present invention relates to inexpensive phytases with low Michaelis constants (abbreviated hereinafter to Km) for phytic acid, which decompose phytic acid as anti-trophic factor contained in feed thereby improving the nutritive values of feed and simultaneously enabling efficient utilization of phosphate released by said decomposition.
Phosphorus is an element essential for every organism. Phosphorus is included in plant-derived feed used in breeding of domestic animals, and 50 to 70% of the phosphorus is present as phytic acid. Phytic acid occurring in a large amount in plant seeds is a major storage substance of phosphate. However, phytic acid is excreted without digestion and absorption in digestive organs in single-stomach animals such as pigs, chickens etc., so its phosphorus is not utilized at all although it is a major storage substance of phosphate. Accordingly, inorganic phosphate is added to feed for single-stomach animals for the purpose of growth promotion. However, addition of phosphate to feed results in an increase in the amount of phosphorus in feces. In recent years, as production of domestic animals is increased, feces from domestic animals are increased to cause an environmental problem in all over the world. In particular, phosphorus contained in feces is mentioned as a cause for the phenomenon of eutrophication in lakes and marshes, and the amount of excreted phosphorus comes to be regulated and there arises the necessity for dealing with it.
Further phytic acid chelates with divalent metals important as nutritious sources, such as magnesium, calcium, zinc, iron etc. to make them hardly adsorbed into animals, resulting in reduction of the nutritive values of feed. Accordingly, phytic acid is considered as an anti-trophic factor.
From the foregoing, improvements in the nutritive values of feed are attempted by treating feed with an enzyme for hydrolyzing phytic acid into inositol and inorganic phosphate thereby permitting the phytic acid to release the phosphate to substitute it for conventionally added phosphate whereby the amount of phosphorus in feces is decreased, and phytic acid as an anti-trophic factor is decomposed [U.S. Pat. No. 3,297,548 (1967), J. Nutrition 101, 1289-1294 (1971)]. Microorganisms known to produce phytase (enzyme decomposing phytic acid) include bacteria such as Bacillus subtilis and Pseudomonas, yeasts such as Saccharomyces cerevisiae, and filamentous fungi such as Aspergillus terreus, Aspergillus ficuum and Aspergillus awamori. With respect to the phytase derived from Aspergillus ficuum, its purification and biochemical properties are described in Preparative Biochem., 18, 443-458 (1988) and its gene and amino acid sequence are described in Gene, 127, 87-94 (1993). With respect to the phytase derived from Aspergillus awamori, its nucleotide sequence and amino acid sequence are described in Gene, 133, 55-62 (1993). 
In order to demonstrate the ability possessed by an enzyme, it is necessary for the concentration of its substrate to be higher than the Michaelis constant (Km), and in the case of enzymes having the same maximum reaction rate (Vmax), an enzyme having a lower Km value does not reduce the reaction rate even at lower substrate concentration as compared with an enzyme having a higher Km value. That is, an enzyme having a lower Km value can maintain the sufficient decomposition rate even at lower substrate concentration, and the amount of the substrate not decomposed can be minimized as compared with an enzyme having a higher Km value.
The Michaelis constants (Km) of known phytases derived from filamentous fungi are 250 xcexcM for Aspergillus ficuum (WO 91/05053) and 330 xcexcM Aspergillus oryzae (Biosci. Biotech. Biochem., 57, 1364-1365 (1993)). 
On one hand, acidic phosphatases are purified from various microorganisms and their properties are reported, and for example, 2 acidic phosphatases derived from Aspergillus ficuum are purified and their properties are examined [Prep. Biochem., 18, 37-65 (1988)]. However, said acidic phosphatases cannot use phytic acid as a substrate, so their utilization for the purpose of improving the nutritive values of feed as described above is not feasible.
Under the circumstances described above, there is a need for phytase which decomposes phytic acid as an anti-trophic factor contained in feed thereby improving the nutritive values of feed and simultaneously enabling efficient utilization of phosphate released by said decomposition.
Accordingly, the object of the present invention is to provide phytases having low Km values for phytic acid and a process for producing said phytases.
As a result of their extensive study for solving the problems described above, from microorganisms belonging to the genus Monascus, the present inventors found novel phytases having Km values of 10 to 110 xcexcM when phytic acid was used as a substrate, and they revealed the properties thereof and stablished a process for producing said phytases to complete the present invention.
That is, the present invention relates to novel phytases having Km values of 10 to 110 xcexcM and a process for producing said phytases.
Specific examples of the novel phytases of the invention include 3 phytases having the following physicochemical properties:
1. Phytase I
1) Km: 27 xcexcM when phytic acid is used as a substrate;
2) optimum pH: pH 5.5;
3) pH stability: stable in the range of pH 5.5 to 6.5;
4) optimum temperature: 50xc2x0 C.;
5) temperature stability: stable up to 35xc2x0 C.;
6) substrate specificity: acting on phytic acid, p-nitrophenylphosphate, D-glucose-6-phosphate, fructose-6-phosphate, D-myo-inositol-2-phosphate, D-myo-inositol-1-phosphate, D-myo-inositol-1,4-diphosphate, and adenosine triphosphate as the substrate;
7) molecular weight: about 80 to 100 kDa (gel filtration method); and
8) isoelectric point: pI 5.7 (chromatofocusing method).
2. Phytase II
1) Km: 20 xcexcM when phytic acid is used as a substrate;
2) optimum pH: pH 6.0;
3) pH stability: stable in the range of pH 6.0 to 7.0;
4) optimum temperature: 50xc2x0 C.;
5) temperature stability: stable up to 50xc2x0 C.;
6) substrate specificity: acting on phytic acid, p-nitrophenylphosphate, D-glucose-6-phosphate, fructose-6-phosphate, D-myo-inositol-2-phosphate, D-myo-inositol-1-phosphate, D-myo-inositol-1,4-diphosphate, and adenosine triphosphate as the substrate;
7) molecular weight: about 120 kDa (gel filtration method); and
8) isoelectric point: pI 4.8 (chromatofocusing method).
3. Phytase III
1) Km: 107 xcexcM when phytic acid is used as a substrate;
2) optimum pH: pH 2.5;
3) pH stability: stable in the range of pH 2.0 to 8.0;
4) optimum temperature: 45xc2x0 C.
5) temperature stability: stable up to 60 xc2x0 C.;
6) substrate specificity: acting on p-nitrophenylphosphate, phytic acid, D-glucose-6-phosphate, fructose-6-phosphate, D-myo-inositol-2-phosphate, D-myo-inositol-1-phosphate, D-myo-inositol-1,4-diphosphate, and adenosine triphosphate as the substrate;
7) molecular weight: about 140 kDa (gel filtration method); and
8) isoelectric point: pI 5.2 (chromatofocusing method); and
9) N-terminal amino acid sequence: shown in SEQ ID NO:1.
The microorganisms used in the present invention may be any microorganisms producing the novel phytases having Km values of 10 to 110 xcexcM when phytic acid is used as a substrate, and examples are microorganisms belonging to the genus Monascus. Specifically, Monascus anka IFO 30873 can be mentioned. Further, animal cells having the ability to produce the novel phytases, which have Km values of 10 to 110 110 xcexcM when phytic acid is used as a substrate, can also be used in the present invention.
The microorganism having the ablity to produce the novel phytase is culture in a conventional culture method until the novel phytase is formed and accumulated, and the novel phytase is recovered from the culture whereby the novel phytase can be produced. Hereinafter, the microorganism or mutant used for producing the novel phytase is called the novel phytase-producing organism.
If the novel phytase-producing organism is a prokaryote such as Escherichia coli or an eukaryote such as filamentous fungus, yeast etc., the medium for culturing said microorganism may be a natural or synthetic medium insofar as the medium contains a carbon source, a nitrogen source, and inorganic salts etc. which can be assimilated by the microorganism and in which the microorganism can be efficiently cultured.
The carbon source may be any one which can be assimilated by the microorganism and includes glucose, fructose, sucrose, molasses containing such sugar, hydrocarbons such as starch, starch hydrolysates etc., organic acids such as acetic acid, propionic acid etc., and alcohols such as ethanol, propanol etc.
The nitrogen source includes ammonia, ammonium salts of various inorganic and organic acids, such ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate etc. and other nitrogenous compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysates, soybean cake, soybean cake hydrolysates, and a wide variety of microorganisms obtained by fermentation and digested materials thereof.
Inorganic materials include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate etc.
Further, a medium containing wheat bran, rice bran etc. as the carbon, nitrogen and inorganic sources supplemented with suitable salts can be used as a medium culturing filamentous fungi.
Culture is conducted under aerobic conditions in shaking culture or in submerged spinner culture under aeration. The culture temperature is preferably 15 to 40xc2x0 C., and the culturing period is usually 16 to 96 hours. The pH during culture is maintained in the range of 3.0 to 9.0. Adjustment of the medium pH is conducted with inorganic or organic acid, alkali solution, urea, calcium carbonate or ammonia.
During culture, antibiotics such as ampicillin, tetracycline etc. may be added to the medium.
If filamentous fungi a re to be cultured in a medium containing solid components such as wheat bran etc., the filamentous fungi are inoculated, mixed sufficiently with the solid components, and spread as a thin layer on a large number of aluminum or stainless steel trays in a cellar and cultured under the controlled conditions of temperature, humidity and ventilation. Specifically, the fungi are subjected to stationary culture in a culture chamber under 100% humidity at 25 to 35xc2x0 C. for 3 to 10 days.
If the novel phytase-producing organism is animal cells, the medium for culturing the animal cells includes generally used RPMI 1640 medium, Eagle""s MEM medium, and mediums containing fetal bovine serum in the above mediums, etc.
Culture is conducted under such conditions as in the presence of 5% CO2 etc. The culture temperature is preferably 35 to 37xc2x0 C., and the culturing period is usually 3 to 7 days.
During culture, antibiotics such as kanamycin, penicillin etc. may be added to the medium.
To isolate and purify the novel phytase from the culture of the novel phytase-producing organism, conventional enzyme isolation and purification methods may be used.
For example, if the novel phytase is accumulated in cells of the novel phytase-producing organism, the cells are collected from the culture by centrifugation, then washed and disrupted by a sonicator, a French press, a Manton-Gaulin homogenizer, a dynomill or the like whereby a cell-free extract is obtained. A supernatant obtained by centrifuging the cell-free extract is subjected to salting-out with e.g. sulfate ammonium, desalting, precipitation with an organic solvent, anion-exchange chromatography on resin such as diethylaminoethyl (DEAE) Sepharose and DIAION HPA-75 (Mitsubishi Chemical Industries Ltd.), cation-exchange chromatography on resin such as S-Sepharose FF (Pharmacia), hydrophobic chromatography on resin such as butyl Sepharose and phenyl Sepharose, gel filtration on molecular sieves, chromatofocusing and electrophoresis such as isoelectric focusing, whereby a purified enzyme preparation can be obtained.
Analysis of the structure of the purified enzyme preparation can be effected by techniques generally used in protein chemistry, for example techniques described in xe2x80x9cProtein Structural Analysis for Gene Cloningxe2x80x9d authored by Hisashi Hirano and published by Tokyo Kagaku Dojin (1993).
If the novel phytase is extracellularly secreted, the culture is subjected to e.g. centrifugation to give a soluble fraction. If solid components such as wheat bran etc. are present in the medium ingredients, the novel phytase is extracted with hot water or the like and subjected to techniques such as centrifugation to give a soluble fraction. From this soluble fraction, a purified enzyme preparation of the novel phytase can be obtained by the same techniques as in isolation and purification from the supernatant of the cell-free extract as described above.
In the present invention, the activity of the novel phytase can be determined according to a standard activity measurement method (see the Reference Example below).
Further, the Km value of the novel phytase can be determined by the Lineweaver-Burk plot in which the activity of the novel phytase, as determined by the standard activity measurement method, is plotted at varying concentrations of the substrate.
The novel phytase of the invention can be utilized in various steps required for converting a salt of phytate into inositol and inorganic phosphate, for example in producing animal feed, soybean processing, liquid feed for pigs and poultry, and inositol or inositol monophosphate from salts of phytate.
Animal feed containing the novel phytase of the invention can be produced by mixing said enzyme with carriers such as wheat chaff, drying the mixture in a spraying column or a fluidized bed, and adding osmotic pressure stabilizers such as sorbitol and preservatives such as benzoic acid to the dried material. The amount of the novel phytase in animal feed is 10 to 5000 U, preferably 100 to 1000 U, per kg of the animal feed.